Prevention and treatment of type-1 diabetes using agonists of the (na++k+)-atpase

ABSTRACT

Methods of preventing and treating type-1 diabetes and its complications using antibody activators that bind to the (Na + +K + )-ATPase.

TECHNICAL FIELD

The invention relates to methods for preventing/treating type-1 diabetes(T1D) and its complications using antibodies (including both endogenousand exogenous) that bind to alpha (α) subunit or beta (β) subunit, orboth, of the (Na⁺+K⁺)-ATPase (NKA) and increase NKA activity. Antibodycapable of increasing NKA activity (activation of NKA) is called NKAagonist or NKA activator antibody. In addition, T1D and itscomplications can also be prevented and treated by administration of anantigenic NKA α or β subunits peptides that induce the production ofendogenous antibodies, which specifically bind to the α or β subunits ofNKA.

BACKGROUND OF INVENTION

T1D known as juvenile diabetes or insulin-dependent diabetes thatdestroys insulin-producing beta (β) cells in the pancreatic islets.Insulin is an important hormone needed to convert sugars, starches andother foods into energy. When too little insulin is given, blood sugarlevels rise. Over time, this can cause dangerous complications.

T1D causes devastating complications that can damage major organs in thebody, including heart, blood vessels, nerves, foot, skin, mouth, eyesand kidneys. Eventually, diabetes complications may be disabling or evenlife-threatening. Clearly, there is an unmet need for prevention andtreatment of T1D and its devastating complications.

NKA is a transmembrane enzyme responsible for the active reciprocaltransport of Na⁺ and K⁺ ions across the plasma membrane of all animalcells. NKA comprises two basic subunits: the α subunit and the βsubunit. The larger α subunit is the functional subunit, which catalyzesthe hydrolysis of ATP for active transport of Na⁺ and K⁺ ions across theplasma membrane; the smaller β subunit does not participate in thecatalytic process of the enzyme, but instead acts as a specificchaperone that assists the biogenesis and correct membrane insertion ofnewly synthesized NKA. The α subunit of NKA has three isoforms includingα1, α2 and α3. The β subunit of NKA also has three isoforms includingβ1, β2 and β3. NKA plays a vitally important role in cell function.Studies have demonstrated that significant reduced NKA activity istightly associated with diabetes-induced complications including kidneyfailure and heart failure, demonstrating that protecting and maintainingNKA functional activity from injury is an essential new target forprevention and treatment of T1D.

BRIEF SUMMARY OF INVENTION

The present invention is based on the surprising discovery that NKAantibody agonists or activators with α or β subunit activation sitebinding specificity can be used to protect insulin-producing β cellfunction, inhibit hyperglycemia, prevent and treat hyperglycemia causedkidney failure, heart failure, protein damages and other dangerouscomplications in a subject. Such NKA activator antibodies thus form thebasis of methods of treating or preventing T1D associated with diseases.

T1D associated diseases involve but not limited to 1) heart and bloodvessel disease and various cardiovascular problems, including coronaryartery disease with chest pain (angina), heart attack, heart failure,stroke, atherosclerosis and high blood pressure; 2) nerve damage(neuropathy) in the walls of the blood vessels (capillaries) thatnourish the nerves, especially in the legs causing tingling, numbness,burning or pain, hyperglycemia, lose sense of feeling in the affectedlimbs, nausea, vomiting, diarrhea or constipation; 3) kidney functiondamage (nephropathy), kidney failure or irreversible end-stage kidneydisease; 4) eye damage, damaged the blood vessels of the retina leadingto diabetic retinopathy, cataracts and glaucoma; 5) various footcomplications; 6) skin and mouth infections and Gum diseases; 7)pregnancy complications may lead to miscarriage, stillbirth, birthdefects, diabetic ketoacidosis, retinopathy, pregnancy-induced highblood pressure and preeclampsia or other disease or condition whereininhibition of T1D would be desirable or necessary.

Examples of NKA activator antibodies having α or β subunit bindingspecificity that can be used in the methods of the present inventioninclude, but are not limited to, SSA78 (also referred as Jianye 2),SSA401 (also referred as KX-2), SSA412 (also referred as KX-1), JY2948,and JY421228 polyclonal, monoclonal, humanized and human versionsthereof, and fragments thereof. These antibodies are capable ofincreasing NKA enzymatic activity (activation of NKA), which aredescribed in Patent Publication No. PCT/US2006/012912 and U.S. Pat. Nos.9,974,842, 9,956,275, 9,790,270, 9,527,923, 9,409,949, 9,416,159,9,238,695, 9,279,020, 9,040,046, 8,945,555, 8,496,929, 8,435,519,8,383,111, 7,754,210, 10,053,505, 10,214,583 and 1,028,736, which areherein incorporated by reference in its entirety for all purposes.

In addition, T1D and its complications can also be treated byadministration of antigenic peptide antigens (or peptide vaccine) of NKAα or β subunits that induce production of endogenous specific antibodyactivators against the α or β subunits of NKA and increase NKA activity.Such peptides and the administration thereof are also described in U.S.Pat. Nos. 9,974,842, 9,956,275, 9,790,270, 9,527,923, 9,409,949,9,416,159, 9,238,695, 9,279,020, 9,040,046, 8,945,555, 8,496,929,8,435,519, 8,383,111, 7,754,210, 10,053,505, 10,214,583 and 10,287,361,which are herein incorporated by reference in its entirety for allpurposes. In a first aspect, the invention thus provides methods forprotecting insulin-generating β cell function comprising contactingpancreatic β cell with an antibody having binding specificity for the αor β subunit of NKA. Exemplary antibodies that may be used in thesemethods include, but are not limited to, (i) antibodies having bindingspecificity for the α or β subunit of NKA, (ii) antibodies havingbinding specificity for one or more of the peptides represented by SEQID NOs:1-7, (iii) NKA activator antibodies in a humanized or humanversions thereof, or a fragment or derivative thereof, and (iv) antigensof SEQ ID NOs: 1-7 to generating endogenous NKA activator antibody, in ahuman or polyclonal versions thereof, or a fragment or derivativethereof. The method may be conducted in vitro or in vivo. The method mayalso be conducted in blood ex vivo.

In a second aspect, the invention provides methods for preventingautoimmune attacks that destroys the β cells in the pancreas comprisingcontacting β cells with an antibody having binding specificity for the αor β subunit of NKA. Exemplary antibodies that may be used in thesemethods include, but are not limited to, (i) antibodies having bindingspecificity for the α or β subunit of NKA, (ii) antibodies havingbinding specificity for one or more of the peptides represented by SEQID NOs:1-7, (iii) NKA activator antibodies in a humanized or humanversions thereof, or a fragment or derivative thereof, and (iv) usingSEQ ID NOs: 1-7 peptide antigen to generate endogenous NKA activatorantibodies, in human or polyclonal versions thereof, or a fragment orderivative thereof. The method may be conducted in vitro or in vivo. Themethod may also be conducted in blood ex vivo.

In a third aspect, the invention provides methods for inhibitinghyperglycemia in a subject comprising administering an effective amountof an antibody having binding specificity for the α or β subunit of NKAto a subject in need thereof. Exemplary antibodies that may be used inthese methods include, but are not limited to, (i) antibodies havingbinding specificity for the α or β subunit of NKA, (ii) antibodieshaving binding specificity for one or more of the peptides representedby SEQ ID NOs:1-7, (iii) NKA activator antibodies in a humanized orhuman versions thereof, or a fragment or derivative thereof, and (iv)using SEQ ID NOs: 1-7 peptide antigen to generate endogenous NKAactivator antibodies, in human or polyclonal versions thereof, or afragment or derivative thereof. The subject may be one that ischaracterized has having or at being at greater risk than the generalpopulation for one or more of the following diseases and conditions: 1)heart and blood vessel disease and various cardiovascular problems,including coronary artery disease with chest pain (angina), heartattack, heart failure, stroke, atherosclerosis and high blood pressure;2) nerve damage (neuropathy) in the walls of the blood vessels(capillaries) that nourish the nerves, especially in the legs causingtingling, numbness, burning or pain, hyperglycemia, lose sense offeeling in the affected limbs, nausea, vomiting, diarrhea orconstipation; 3) kidney function damage (nephropathy), kidney failure orirreversible end-stage kidney disease; 4) eye damage, damaged the bloodvessels of the retina leading to diabetic retinopathy, cataracts andglaucoma; 5) various foot complications; 6) skin and mouth infectionsand Gum diseases; 7) pregnancy complications may lead to miscarriage,stillbirth, birth defects, diabetic ketoacidosis, retinopathy,pregnancy-induced high blood pressure and preeclampsia or other diseaseor condition wherein inhibition of T1D would be desirable or necessary.

In a fourth aspect, the invention provides methods for protecting kidneyfunction and preventing diabetic kidney failure in a subject comprisingadministering an effective amount of an antibody having bindingspecificity for the α or β subunit of NKA to a subject in need thereof.Exemplary antibodies that may be used in these methods include, but arenot limited to, (i) antibodies having binding specificity for the α or βsubunit of NKA, (ii) antibodies having binding specificity for one ormore of the peptides represented by SEQ ID NOs:1-5, (iii) NKA activatorantibodies in a humanized or human versions thereof, or a fragment orderivative thereof, and (iv) using SEQ ID NOs: 1-5 peptide antigen togenerate endogenous NKA activator antibodies, in human or polyclonalversions thereof, or a fragment or derivative thereof. The subject mayone that is at greater risk than the general population for kidneyfailure. The subject may be one that is characterized has having or atbeing at greater risk than the general population for one or more of thefollowing diseases and conditions: diabetic nephropathy, kidneynecrosis, kidney failure, irreversible end-stage kidney disease, whichrequires dialysis or a kidney transplant.

In a fifth aspect, the invention provides methods for treating T1Dinduced hyperglycemia and complications in a subject comprisingadministering an effective amount of an antibody having bindingspecificity for the α subunit of NKA to a subject in need thereof.Exemplary antibodies that may be used in these methods include, but arenot limited to, (i) antibodies having binding specificity for the αsubunit of NKA, (ii) antibodies having binding specificity for one ormore of the peptides antigen or peptide vaccine represented by SEQ IDNOs:1-7, (iii) NKA activator antibodies in a humanized or human versionsthereof, or a fragment or derivative thereof, and (iv) using SEQ ID NOs:1-7 peptide antigen to generate endogenous NKA activator antibodies, inhuman or polyclonal versions thereof, or a fragment or derivativethereof. The subject may be one that is characterized has having or atbeing at greater risk than the general population for one or more of thefollowing diseases and conditions: 1) heart and blood vessel disease andvarious cardiovascular problems, including coronary artery disease withchest pain (angina), heart attack, heart failure, stroke,atherosclerosis and high blood pressure; 2) nerve damage (neuropathy) inthe walls of the blood vessels (capillaries) that nourish the nerves,especially in the legs causing tingling, numbness, burning or pain,hyperglycemia, lose sense of feeling in the affected limbs, nausea,vomiting, diarrhea or constipation; 3) kidney function damage(nephropathy), kidney failure or irreversible end-stage kidney disease;4) eye damage, damaged the blood vessels of the retina leading todiabetic retinopathy, cataracts and glaucoma; 5) various footcomplications; 6) skin and mouth infections and Gum diseases; 7)pregnancy complications may lead to miscarriage, stillbirth, birthdefects, diabetic ketoacidosis, retinopathy, pregnancy-induced highblood pressure and preeclampsia or other disease or condition whereininhibition of T1D would be desirable or necessary.

In a sixth aspect, the invention provides methods for treatingautoimmune attacks that destroys the β cells in the pancreas in asubject comprising administering an effective amount of an antibodyhaving binding specificity for the α or β subunit of NKA to a subject inneed thereof. Exemplary antibodies that may be used in these methodsinclude, but are not limited to, (i) antibodies having bindingspecificity for the α or β subunit of NKA, (ii) antibodies havingbinding specificity for one or more of the peptides represented by SEQID NOs:1-7, (iii) NKA activator antibodies in a humanized or humanversions thereof, or a fragment or derivative thereof, and (iv) usingSEQ ID NOs: 1-7 peptide antigen to generate endogenous NKA activatorantibodies, in human or polyclonal versions thereof, or a fragment orderivative thereof. The subject may be one that is characterized hashaving or at being at greater risk than the general population for oneor more of the following diseases and conditions: 1) heart and bloodvessel disease and various cardiovascular problems, including coronaryartery disease with chest pain (angina), heart attack, heart failure,stroke, atherosclerosis and high blood pressure; 2) nerve damage(neuropathy) in the walls of the blood vessels (capillaries) thatnourish the nerves, especially in the legs causing tingling, numbness,burning or pain, hyperglycemia, lose sense of feeling in the affectedlimbs, nausea, vomiting, diarrhea or constipation; 3) kidney functiondamage (nephropathy), kidney failure or irreversible end-stage kidneydisease; 4) eye damage, damaged the blood vessels of the retina leadingto diabetic retinopathy, cataracts and glaucoma; 5) various footcomplications; 6) skin and mouth infections and Gum diseases; 7)pregnancy complications may lead to miscarriage, stillbirth, birthdefects, diabetic ketoacidosis, retinopathy, pregnancy-induced highblood pressure and preeclampsia or other disease or condition whereininhibition of T1D would be desirable or necessary.

In a seventh aspect, the invention provides methods for preventing T1Dinduced heart failure and kidney failure in a subject comprisingadministering an effective amount of an antibody having bindingspecificity for the α or β subunit of NKA to a subject in need thereof.Exemplary antibodies that may be used in these methods include, but arenot limited to, (i) antibodies having binding specificity for the α or βsubunit of NKA, (ii) antibodies having binding specificity for one ormore of the peptides represented by SEQ ID NOs:1-7, (iii) NKA activatorantibodies in a humanized or human versions thereof, or a fragment orderivative thereof, and (iv) using SEQ ID NOs: 1-7 peptide antigen togenerate endogenous NKA activator antibodies, in human or polyclonal ormonoclonal versions thereof, or a fragment or derivative thereof.Exemplary diseases include, but are not limited to, heart and bloodvessel disease and various cardiovascular problems, including coronaryartery disease with chest pain (angina), heart failure, heart attack,stroke, atherosclerosis and high blood pressure; diabetic nephropathy,kidney failure or irreversible end-stage kidney disease, which requiresdialysis or a kidney transplant.

In an eighth aspect, the invention provides methods for prolonging lifespan for T1D patient comprising administering an effective amount of anantibody having binding specificity for the α or β subunit of NKA to asubject in need thereof. Exemplary antibodies that may be used in thesemethods include, but are not limited to, (i) antibodies having bindingspecificity for the α or β subunit of NKA, (ii) antibodies havingbinding specificity for one or more of the peptides represented by SEQID NOs:1-7, (iii) NKA activator antibodies in a humanized or humanversions thereof, or a fragment or derivative thereof, and (iv) usingSEQ ID NOs: 1-7 peptide antigen to generate endogenous NKA activatorantibodies, in human or polyclonal or monoclonal versions thereof, or afragment or derivative thereof.

In each of these aspects, the antibody may be in a pharmaceuticalformulation comprising the antibody and a pharmaceutically acceptablecarrier.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedherein, which form the subject matter of the claims of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E & FIG. 1F).Representative slide of pancreas that houses pancreatic islet fromnondiabetic control mice and type-1 diabetes NOD female mice treatedwith different NKA antibody activators. These islets contain beta cells,which produce insulin. Specific NKA antibody activator was generatedwithin NOD mice body by injecting specific antibody peptide antigen.Generation of SSA412 in NOD mice was determined by ELISA Pancreases wereisolated and fixed in formaldehyde, embedded in paraffin, sectioned, andstained with hematoxylin and eosin (H&E). FIG. 1A: Normal histologicalappearance of a pancreatic islet in nondiabetic mice control group. FIG.1B: NOD mice treated with antibody SSA412. FIG. 1C: Treated withantibody SSA401. FIG. 1D: Treated with antibody SSA78. FIG. 1E: Treatedwith antibody JY2948. FIG. 1F: Treated with antibody JY421228. All micewere 20-month old. Experimental results show healthy insulin-producingislets in NKA antibody activator-treated NOD mice and demonstrate aprotective effect of NKA antibody activators on the structure andfunction of pancreatic beta cells. Original magnification: ×200.

FIG. 2. Serum insulin concentration, as determined by Millipore insulinassay RIA-Kit, in NOD female mice in nondiabetic (n=5), untreateddiabetic (n=10) and treated with SSA412 (n=10) groups. The NKA antibodyactivator SSA412 was generated by injecting specific SSA412antibody-generating peptide antigen into NOD mice. Generation of SSA412in NOD mice was determined by ELISA. Experimental results demonstratethat NKA antibody activator SSA412 protects beta cell function andpromote beta cell insulin generation. Experiment data are expressed asmean±SE. The statistical significant difference between treated anduntreated groups (p<0.001).

FIG. 3. Blood glucose of NOD female mice at 4-month of age innondiabetic, untreated diabetic and treated with NKA activator SSA412groups. The NKA antibody activator SSA412 was generated by injectingspecific SSA412 antibody-generating peptide antigen into NOD mice.Generation of SSA412 in NOD mice was determined by ELISA. Experimentresults show that both nondiabetic mice (left, n=5) and NKA activatorSSA412-treated NOD mice (right, n=15) have normal blood glucose level.In contrast, hyperglycemia was developed in untreated NOD mice group(middle, n=15). The results demonstrate the therapeutic efficacy of NKAantibody activator-based immunotherapy on preventing hyperglycemia intype-1 diabetes female NOD mice. Experiment data are expressed asmean±SE. There is a statistically significant difference betweenuntreated and treated NOD mice (p<0.001).

FIG. 4. Kaplan-Meier survival analysis for NOD female mice. Mice weretreated with NKA antibody activator SSA412 (n=12) or without treatment(n=12). There is a statistically significant difference between survivalcurves (p<0.001). The group of mice treated with NKA antibody activatorSSA412 demonstrated statistically significant longer survival thanuntreated group.

FIG. 5. Kidney morphological comparison among six-month-old female NODmice. The NKA antibody activator SSA412 was generated by injectingspecific SSA412 antibody-generating peptide antigen into NOD mice.Generation of SSA412 in NOD mice was determined by ELISA. Left: Healthynondiabetic mice control. Middle: NOD mice treated with NKA antibodyactivator SSA412. Right: Untreated NOD mice kidney. The results show theefficacy of NKA antibody activator-based immunotherapy, which protectedkidney function and prevented kidney enlargement and failure (n=10).

FIG. 6 (FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E &FIG. 6F).Histopathology comparison of kidney tissues under untreated and treatedwith NKA activator-based immunotherapy conditions. The NKA antibodyactivator SSA412 was generated by injecting specific SSA412antibody-generating peptide antigen into NOD mice. Generation of SSA412in NOD mice was determined by ELISA. FIG. 6A: Section of nondiabetickidney tissue showing normal appearance of glomerulus (×400). FIG. 6B:Section of untreated NOD female mice diabetic kidney tissue showingglomerular focal necrosis (×400). FIG. 6C: Section of immunotherapytreated NOD female mice showing normal glomerulus (×400). FIG. 6D:Section of nondiabetic kidney tissue showing normal appearance oftubules (×200). FIG. 6E: Section of untreated NOD female mice diabetickidney tissue showing tubular necrosis and degeneration (×200). FIG. 6F:Section of immunotherapy treated NOD female mice showing normal kidneytubules (×200). These data provide evidence to demonstrate that NKAactivator-based immunotherapy protected kidney function against type-1diabetes-induced kidney necrosis and failure in female NOD mice. (n=6).

FIG. 7. NKA activator-based immunotherapy protects kidney function andprevent T1D induced kidney failure complications in NOD mice. The NKAantibody activator SSA412 was generated by injecting specific SSA412antibody-generating peptide antigen into NOD mice. Generation of SSA412in NOD mice was determined by ELISA. Serum was separated from the bloodand collected for creatinine testing. Experimental results demonstratedthat the NKA antibody activator-based immunotherapy protected kidneyfunction by maintaining a normal creatinine concentration in blood(n=15). Left: Nondiabetic. Middle: Untreated. Right: Treated withSSA412. Experiment data are expressed as mean±SE. There is astatistically significant difference between untreated and treated NODmice (p<0.001).

FIG. 8. NKA activator-based immunotherapy protects kidney NKA functionand prevents T1D induced inactivation of enzyme in NOD mice. The NKAantibody activator SSA412 was generated by injecting specific SSA412antibody-generating peptide antigen into NOD mice. Generation of SSA412in NOD mice was determined by ELISA. Homogenates of NOD kidney tissueunder different experimental conditions were prepared and anouabain-sensitive ATPase assay was performed to determine the catalyticactivity of NKA enzyme. Left: Nondiabetic kidney control tissue. Middle:diabetic kidney failure tissue. Right: Kidney treated with SSA412.Experiment data are expressed as mean±SE. There is a statisticallysignificant difference between untreated and treated NOD mice (p<0.001).The results show that NKA antibody activator-based immunotherapyprotected NKA activity while NKA activity was significantly impaired inuntreated diabetic kidney (n=15).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

As used herein, “a” or “an” may mean one or more. As used herein whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more. Furthermore, unless otherwise required bycontext, singular terms include pluralities and plural terms include thesingular.

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

As outlined in a general manner above, the present invention is based onthe surprising discovery that NKA antibody agonists or activators with αor β subunit activation site binding specificity can be used to protectinsulin-producing β cell function, inhibit hyperglycemia, prevent andtreat hyperglycemia caused kidney failure, heart failure, proteindamages and other dangerous complications in a subject. Such NKAactivator antibodies thus form the basis of methods of treating orpreventing T1D associated with diseases, whether in vitro or in vivo, toinhibit, treat, and prevent T1D and its complications in a subject. Theantibodies and their peptide antigens also form the basis of methods oftreating or preventing T1D and T1D associated diseases andcomplications.

Antibodies

The skilled artisan will understand that the particular attributes ofthe antibodies that may be used in the methods of the present inventionare only confined by (i) the ability to bind with specificity to the αor β subunit of NKA, and (ii) the ability to inhibit T1D and itscomplications

As described in PCT/US2006/012912 and U.S. Pat. Nos. 9,974,842,9,956,275, 9,790,270, 9,527,923, 9,409,949, 9,416,159, 9,238,695,9,279,020, 9,040,046, 8,945,555, 8,496,929, 8,435,519, 8,383,111,7,754,210, 10,053,505, 10,214,583 and 10,287,361, seven antibodies havebeen prepared that specifically bind the α or β subunit of NKA. Thefollowing antibodies specifically bind to the α subunit of NKA, namelyantibody SSA95 (also referred as Jianye 1 antibody), SSA97 (alsoreferred as Zulan antibody), SSA78 (also referred as Jianye 2 antibody),SSA401 (also referred as KX-2 antibody), and SSA412 (also referred asKX-1 antibody). Antibodies JY2948 and JY421228 specifically bind to theβ subunit of NKA. As shown in the Examples below, these antibodies maybe used in the methods of the present invention. Antibody SSA95 binds toamino acids KRQPRNPKTDKLVNE (SEQ ID NO:1), SSA97 binds to amino acidsVPAISLAYEQAESD (SEQ ID NO:2), SSA78 binds to amino acids RSATEEEPPNDD(SEQ ID NO:3), SSA401 binds to amino acids HLLGIRETWDDRWIN (SEQ IDNO:4), SSA412 binds to amino acids DVEDSYGQQWTYEQR (SEQ ID NO:5), JY2948binds to amino acids KERGEFNHERGER (SEQ ID NO:6), and JY421228 binds toamino acids RDEDKDKVGNIEY (SEQ ID NO:7). The invention thereforeprovides the use of NKA activator antibodies SSA95, SSA97, SSA78,SSA401, SSA412, JY2948, and antibody JY421228 in the methods disclosedherein.

The invention also provides the use of antibodies that specifically bindan epitope of the α or β subunit of NKA comprising the amino acidsequence KRQPRNPKTDKLVNE (SEQ ID NO:1), VPAISLAYEQAESD (SEQ ID NO:2),RSATEEEPPNDD (SEQ ID NO:3), HLLGIRETWDDRWIN (SEQ ID NO:4),DVEDSYGQQWTYEQR (SEQ ID NO:5), KERGEFNHERGER (SEQ ID NO:6), andRDEDKDKVGNIEY (SEQ ID NO:7), or any combination thereof.

The invention further provides for the use of antibodies having bindingspecificity for an epitope of the α or β subunit of NKA comprising theamino acid SEQ ID NOs: 1-7. Antibody SSA95 binds to amino acidsKRQPRNPKTDKLVNE (SEQ ID NO:1), SSA97 binds to amino acids VPAISLAYEQAESD(SEQ ID NO:2), SSA78 binds to amino acids RSATEEEPPNDD (SEQ ID NO:3),SSA401 binds to amino acids HLLGIRETWDDRWIN (SEQ ID NO:4), SSA412 bindsto amino acids DVEDSYGQQWTYEQR (SEQ ID NO:5), JY2948 binds to aminoacids KERGEFNHERGER (SEQ ID NO:6), and JY421228 binds to amino acidsRDEDKDKVGNIEY (SEQ ID NO:7).

The invention further provides for the use of antibodies having bindingspecificity for variants of each of the peptides of SEQ ID NOs:1-7, thevariants having 8 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 orfewer, or 1 amino acid change in comparison to the peptides of SEQ IDNOs:1-5. The changes are each individually selected from insertions,deletions and substitutions. The substitutions may be conservative ornon-conservative amino acid substitutions. Each of the variant peptidesmaintains the ability to induce production of antibodies thatspecifically bind the α or β subunit of NKA and that have the ability toinhibit T1D and T1D complications.

In addition, the invention provides for the use of antibodies havingbinding specificity for other epitopes of the α and β subunit of NKA,with those antibodies having binding specificity for other epitopes ofthe α or β subunit of NKA being of particular note.

The antibodies used in the methods of the present invention and definedabove may be polyclonal, monoclonal, humanized, chimeric antibodies, orhuman version, and the antibodies may be in the form of an antiserumcomprising the antibodies. The antibodies may be of any class, such asIgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE. The antibodies maybe isolated antibodies, purified antibodies, exogenous antibodies,endogenous antibodies, single chain antibodies, single-chain variablefragment, or a combination thereof.

The antibodies may also be antibody fragments of less than the entireantibody, including, but not limited to, single chain antibodies,F(ab′)2 fragments, Fab fragments, and fragments produced by an Fabexpression library, and derivatives of the antibodies and fragmentsdefined herein, with the only limitation being that the antibodyfragments and derivatives retain the ability to bind the α or β subunitand inhibit T1D and its complications. It will thus be clear to theskilled artisan that all references to “antibodies” herein include bothfull-size antibodies as well as antibody fragments, as defined herein.

The antibodies may be produced in any species of animal, thoughpreferably from a mammal such as a human, simian, mouse, rat, rabbit,guinea pig, horse, cow, sheep, goat, pig, dog or cat. For example, theantibodies can be human antibodies or humanized antibodies, or anyantibody preparation suitable for administration to a human. For theproduction of the antibodies, the selected species of animal can beimmunized by injection with one or more of the peptides or variantsdiscussed herein. The peptides and variants may be administered inconjunction with one or more pharmaceutically acceptable adjuvants toincrease the immunological response. Suitable adjuvants include, but arenot limited to, Freund's Complete and Incomplete Adjuvant, Titermax, Oilin Water Adjuvants, as well as Aluminum compounds where antigens,normally peptides, are physically precipitated with hydrated insolublesalts of aluminum hydroxide or aluminum phosphate. Other adjuvantsinclude liposome-type adjuvants comprising spheres having phospholipidbilayers that form an aqueous compartment containing the peptide andprotect it from rapid degradation, and that provide a depot effect forsustained release. Surface active agents may also be used as adjuvantsand include lipoteichoic acid of gram-positive organisms, lipid A, andTDM. Quil A and QS-21 (saponin-type adjuvants), monophosphoryl lipid A,and lipophilic MDP derivatives are suitable adjuvants that havehydrophilic and hydrophobic domains from which their surface-activeproperties arise. Compounds normally found in the body such as vitamin Aand E, and lysolecithin may also be used as surface-active agents. Otherclasses of adjuvants include glycan analog, coenzyme Q, amphotericin B,dimethyldioctadecylammonium bromide (DDA), levamisole, and benzimidazolecompounds. The immunostimulation provided by a surface active agent mayalso be accomplished by either developing a fusion protein withnon-active portions of the cholera toxin, exotoxin A, or the heat labiletoxin from E. coli. Immunomodulation through the use of anti-IL-17, antiIFN-γ, anti-IL-12, IL-2, IL-10, or IL-4 may also be used to promote astrong Th2 or antibody mediated response to the immunogenic formulation.

Means for preparing antibodies are very well known in the art. Theantibodies of the invention can be prepared using any known techniquethat provides for the production of antibody molecules. Suitabletechniques include, but are not limited to, the hybridoma techniqueoriginally described by Koehler and Milstein (Nature 256:495-497(1975)), the human B-cell hybridoma technique (Kosbor et al., ImmunolToday 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030(1983)), and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96(1985)). Each of these publications is herein incorporated by referencein its entirety. Additionally, antibodies can be produced by inducing invivo production in the lymphocyte population or by screening recombinantimmunoglobulin libraries or panels of highly specific binding reagentsas disclosed in Orlandi et al., Proc Natl. Acad. Sci. USA 86: 3833-3837(1989), and in Winter G. and Milstein C., Nature 349:293-299 (1991),both of which is herein incorporated by reference in its entirety.

Humanized antibodies are those where a human antibody has beenengineered to contain non-human complementarity-determining regions(CDRs) derived from an antibody produced in a non-human host against aselected antigen. Means for producing humanized antibodies arewell-known in the art and include Vaswani S K, and Hamilton R G, AnnAllergy Asthma Immunol. 81(2):105-15 (1998) and Kashmiri S V et al.,Methods 36 (1):25-34 (2005), each of which is herein incorporated byreference in its entirety.

Human antibodies are those where an antibody produced in a non-humanhost against a selected antigen. Means for producing human antibodiesare well-known in the art and include Bruggemann M et al., Arch ImmunolTher Exp (Warsz). 63(2):101-108 (2014), Pruzina S et al., Protein EngDes Sel. 24 (10):791-799 (2011), and Smith K et al., Nature. 4:372-384(2009), each of which is herein incorporated by reference in itsentirety.

Chimeric antibodies are those where an antigen binding region (e.g.,F(ab′)₂ or hypervariable region) of a non-human antibody is transferredinto the framework of a human antibody by recombinant DNA techniques.Techniques developed for the production of such antibodies include thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity.Such techniques are also well known and include: Morrison et al., ProcNatl. Acad. Sci 81:6851-6855 (1984); Neuberger et al., Nature312:604-608(1984); Takeda et al., Nature 314:452-454(1985), each ofwhich is herein incorporated by reference in its entirety.

Techniques for the production of single chain antibodies are describedin in U.S. Pat. No. 4,946,778, incorporated herein by reference in itsentirety.

Antibody fragments such as F(ab′)2 fragments can be produced by pepsindigestion of the antibody molecule, and Fab fragments can be generatedby reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries can be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse W. D. et al., Science 256:1275-1281 (1989),herein incorporated by reference in its entirety).

The invention provides for the use of pharmaceutical formulationscomprising one or more of the antibodies of the invention and apharmaceutically acceptable carrier. Such formulations may beadministered to a subject when practicing the methods of the presentinvention. Suitable examples of carriers are well known to those skilledin the art and include water, water-for-injection, saline, bufferedsaline, dextrose, glycerol, ethanol, propylene glycol, polysorbate 80(Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400),PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188,hydrophilic and hydrophobic carriers, and combinations thereof.Hydrophobic carriers include, for example, fat emulsions, lipids,PEGylated phospholipids, polymer matrices, biocompatible polymers,lipospheres, vesicles, particles, and liposomes. The terms specificallyexclude cell culture medium. The formulations may further comprisestabilizing agents, buffers, antioxidants and preservatives, tonicityagents, bulking agents, emulsifiers, suspending or viscosity agents,inert diluents, fillers, and combinations thereof.

The identity of the carrier(s) will also depend on the means used toadminister pharmaceutical formulations comprising antibodies to asubject. For example, pharmaceutical formulations for intramuscularpreparations can be prepared where the carrier is water-for-injection,0.9% saline, or 5% glucose solution. Pharmaceutical formulations mayalso be prepared as liquid or powdered atomized dispersions for deliveryby inhalation. Such dispersion typically contain carriers common foratomized or aerosolized dispersions, such as buffered saline and/orother compounds well known to those of skill in the art. The delivery ofthe pharmaceutical formulations via inhalation has the effect of rapidlydispersing the vaccine formulation to a large area of mucosal tissues aswell as quick absorption by the blood for circulation. One example of amethod of preparing an atomized dispersion is described in U.S. Pat. No.6,187,344, entitled, “Powdered Pharmaceutical Formulations HavingImproved Dispersibility,” which is hereby incorporated by reference inits entirety.

Additionally, the pharmaceutical formulations may also be administeredin a liquid form. The liquid can be for oral dosage, for ophthalmic ornasal dosage as drops, or for use as an enema or douche. When thepharmaceutical formulation is formulated as a liquid, the liquid can beeither a solution or a suspension of the pharmaceutical formulation.There is a variety of suitable formulations for the solution orsuspension of the pharmaceutical formulations that are well known tothose of skill in the art, depending on the intended use thereof. Liquidformulations for oral administration prepared in water or other aqueousvehicles may contain various suspending agents such as methylcellulose,alginates, tragacanth, pectin, kelgin, carrageenan, acacia,polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations mayalso include solutions, emulsions, syrups and elixirs containing,together with the active compound(s), wetting agents, sweeteners, andcoloring and flavoring agents.

The invention also includes inhibiting T1D and its complications byadministering a peptide vaccine that includes one or more peptidesselected from a group of peptides having the amino acid sequencesKRQPRNPKTDKLVNE (SEQ ID NO:1), VPAISLAYEQAESD (SEQ ID NO:2),RSATEEEPPNDD (SEQ ID NO:3), HLLGIRETWDDRWIN (SEQ ID NO:4),DVEDSYGQQWTYEQR (SEQ ID NO:5), KERGEFNHERGER (SEQ ID NO:6), andRDEDKDKVGNIEY (SEQ ID NO:7), and variants or fragments thereof. Thesepeptides represent antigenic determinants or epitopes on the α or βsubunit of NKA. The peptide vaccines stimulate the host immune system togenerate antibodies against the respective one or more peptide epitopes.Methods of using peptide antigen for making, isolating and purifying NKAactivity-increasing antibodies and the above-identified peptideantigenic determinants are described in U.S. Patent Applications20040057956 and 20030228315, the entire contents of which are herebyincorporated by reference as if fully set forth herein.

Methods for Inhibiting T1D

As indicated above, the present invention includes methods forinhibiting T1D and its complications. This method comprising contactingpancreatic β cells with an antibody having binding specificity for the αor β subunit of NKA. It will be apparent to the skilled artisan thatthis method can be practice in vitro, in vivo and ex vivo.

Any of the antibodies described herein, whether polyclonal ormonoclonal, can be used in the method, as well as humanized or chimericversions or human versions of the antibodies, and fragments of any ofthese. Exemplary antibodies that may be used in these methods include,but are not limited to, (i) antibodies having binding specificity forthe α or β subunit of NKA, (ii) antibodies having binding specificityfor one or more of the peptides represented by SEQ ID NOs:1-7, (iii)antibodies in polyclonal, monoclonal, humanized and human versionsthereof, and (iv) antibodies in an exogenous or endogenous versionsthereof.

Methods for Inhibiting T1D Induced Complications

The present invention includes methods for inhibiting T1D complications.This method comprising contacting pancreatic β cells with an antibodyhaving binding specificity for the α or β subunit of NKA. It will beapparent to the skilled artisan that this method can be practice invitro, in vivo and ex vivo.

Any of the antibodies described herein, whether polyclonal ormonoclonal, can be used in the method, as well as humanized or chimericversions of the antibodies, and fragments of any of these. Exemplaryantibodies that may be used in these methods include, but are notlimited to, (i) antibodies having binding specificity for the α or βsubunit of NKA, (ii) antibodies having binding specificity for one ormore of the peptides represented by SEQ ID NOs:1-7, (iii) antibodiespolyclonal, monoclonal, humanized and human versions thereof, and (iv)antibodies in an exogenous or endogenous versions thereof.

Methods of Treatment

The invention also provides methods for treating or preventingparticular diseases, disorders and conditions in a subject by inhibitingT1D.

The invention thus includes methods for inhibiting T1D complications ina subject comprising administering an effective amount of an antibodyhaving binding specificity for the α or β subunit of NKA to a subject inneed thereof. While the subject is not limited to one having aparticular disease or condition, the subject may be one that ischaracterized has having or at being at greater risk than the generalpopulation for one or more of the following diseases and conditions: 1)heart and blood vessel disease and various cardiovascular problems,including coronary artery disease with chest pain (angina), heartattack, heart failure, stroke, atherosclerosis and high blood pressure;2) nerve damage (neuropathy) in the walls of the blood vessels(capillaries) that nourish the nerves, especially in the legs causingtingling, numbness, burning or pain, hyperglycemia, lose sense offeeling in the affected limbs, nausea, vomiting, diarrhea orconstipation; 3) kidney function damage (nephropathy), kidney failure orirreversible end-stage kidney disease; 4) eye damage, damaged the bloodvessels of the retina leading to diabetic retinopathy, cataracts andglaucoma; 5) various foot complications; 6) skin and mouth infectionsand Gum diseases; 7) pregnancy complications may lead to miscarriage,stillbirth, birth defects, diabetic ketoacidosis, retinopathy,pregnancy-induced high blood pressure and preeclampsia or other diseaseor condition wherein inhibition of T1D would be desirable or necessary.

The invention includes methods for inhibiting, treating or preventingT1D and T1D complications in a subject, where the method comprisesadministering an effective amount of an antibody having bindingspecificity for the α or β subunit of NKA to a subject in need thereof.While the subject is not limited to one having a particular disease orcondition, the subject may be one that is characterized has having or atbeing at greater risk than the general population for one or more of thefollowing diseases and conditions: 1) heart and blood vessel disease andvarious cardiovascular problems, including coronary artery disease withchest pain (angina), heart attack, heart failure, stroke,atherosclerosis and high blood pressure; 2) nerve damage (neuropathy) inthe walls of the blood vessels (capillaries) that nourish the nerves,especially in the legs causing tingling, numbness, burning or pain,hyperglycemia, lose sense of feeling in the affected limbs, nausea,vomiting, diarrhea or constipation; 3) kidney function damage(nephropathy), kidney failure or irreversible end-stage kidney disease,which requires dialysis or a kidney transplant; 4) eye damage, damagedthe blood vessels of the retina leading to diabetic retinopathy,cataracts and glaucoma; 5) various foot complications; 6) skin and mouthinfections and Gum diseases; 7) pregnancy complications may lead tomiscarriage, stillbirth, birth defects, diabetic ketoacidosis,retinopathy, pregnancy-induced high blood pressure and preeclampsia orother disease or condition wherein inhibition of T1D would be desirableor necessary.

Any of the antibodies described herein, whether polyclonal ormonoclonal, can be used in the method, as well as humanized or chimericor human versions of the antibodies, and fragments and derivatives ofany of these. Exemplary antibodies that may be used in these methodsinclude, but are not limited to, (i) antibodies having bindingspecificity for the α or β subunit of NKA, including isoform of α and βsubunit, (ii) antibodies having binding specificity for one or more ofthe peptides represented by SEQ ID NOs:1-7, (iii) antibodies in apolyclonal, monoclonal, humanized or human versions thereof, or afragment, or single-chain antibody, or single-chain variable fragment,or derivative thereof, and (iv) antibodies in an exogenous or endogenousversions thereof, or a fragment or derivative thereof. The antibody maybe administered as a pharmaceutical formulation comprising the antibodyand a pharmaceutically acceptable carrier.

As used herein, the terms “treat”, “treating” and “treatment” have theirordinary and customary meanings, and include one or more of,ameliorating T1D, ameliorating a symptom of T1D complications, ordecreasing in severity and/or frequency a symptom of T1D complications.Treatment means ameliorating or decreasing by about 1% to about 100%versus a subject to which the antibody has not been administered.Preferably, the ameliorating or decreasing or inhibiting is about 100%,about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%,about 10%, about 5% or about 1%. The treatment may begin prior to,concurrent with, or after the onset of clinical symptoms of T1D and T1Dcomplications. The results of the treatment may be permanent or maycontinue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days),weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6or more months).

As used herein, the terms “prevent”, “preventing” and “prevention” havetheir ordinary and customary meanings, and include one or more of,stopping, averting, avoiding or blocking T1D and its complications, theoccurrence of a symptom of T1D and its complications, the recurrence ofa symptom of T1D and its complications, the development of T1D and itscomplications or the progression of T1D and its complications.Prevention means stopping by at least about 95% versus a subject towhich the antibody has not been administered. Preferably, the stoppingis about 100%, about 99%, about 98%, about 97%, about 96% or about 95%.The results of the prevention may be permanent or may continue for aperiod of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1,2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6 or more months).

As used herein, the terms “inhibit”, “inhibiting” and “inhibition” havetheir ordinary and customary meanings, and include one or more of,hindering, impeding, obstructing, deterring or restraining T1D and itscomplications, the occurrence of a symptom of T1D and its complications,the recurrence of a symptom of T1D and its complications, thedevelopment of T1D and its complications, or the progression of T1D andits complications. Inhibition means impeding by about 1% to about 100%versus a subject to which the antibody has not been administered.Preferably, the impeding is about 100%, about 99%, about 98%, about 97%,about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about50%, about 40%, about 30%, about 20%, about 10%, about 5% or about 1%.The course of therapy may begin prior to, concurrent with, or after theonset of clinical symptoms of T1D and its complications. Thus, thesubject may have T1D and its complications, or merely be susceptible toT1D and its complications. The results of the inhibition may bepermanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6or 7 days), weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2,3, 4, 5, 6 or more months).

The antibodies and formulations may be administered to a subject usingdifferent schedules, depending on the particular aim or goal of themethod; the age and size of the subject; and the general health of thesubject, to name only a few factors to be considered. In general, theantibodies and formulations may be administered once, or twice, threetimes, four times, five times, six times or more, over a course oftreatment, inhibition or prevention. The timing between each dose in adosing schedule may range between days, weeks, months, or years, anincludes administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30or more weeks. The same quantity of antibody may be administered in eachdose of the dosing schedule, or the amounts in each dose may vary. Theidentity of the particular antibody may also vary or remain the same ineach dose in a dosing schedule.

In each of the methods of the present invention, an “effective amount”of an antibody or a pharmaceutical formulation comprising an antibody isadministered to a subject. The effective amount will vary betweensubjects. However, the effective amount is one that is sufficient toachieve the aim or goal of the method, whether inhibiting, treating orpreventing. As an example, an effective amount of an antibody used inthe methods of the invention is typically between about 0.1 μg to about1000 μg of antibody per kg of body weight of the subject to which theantibody is administered. An effective amount also includes betweenabout 1 μg to about 500 μg, between about 10 μg to about 200 μg, betweenabout 1 μg to about 800 μg, between about 10 μg to about 800 μg, betweenabout 1 μg to about 300 μg, and between about 10 μg to about 300 μg ofantibody per kg of body weight of the subject.

Appropriate doses and dosing schedules can readily be determined bytechniques well known to those of ordinary skill in the art withoutundue experimentation. Such a determination will be based, in part, onthe tolerability and efficacy of a particular dose.

Administration of the antibody or formulation may be via any of themeans commonly known in the art of antibody delivery. Such routesinclude intravenous, intraperitoneal, intramuscular, subcutaneous andintradermal routes of administration, as well as nasal application, byinhalation, ophthalmically, orally, rectally, vaginally, or by any othermode that results in the antibody or formulation contacting mucosaltissues.

The term “subject” is intended to mean an animal, such birds or mammals,including humans and animals of veterinary or agricultural importance,such as dogs, cats, horses, sheep, goats, and cattle.

A kit comprising the necessary components for practicing the methods ofthe invention, including an antibody or a pharmaceutical formulationcomprising an antibody, and instructions for its use is also within thepurview of the present invention.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All documents, papers and published materials referenced herein,including books, journal articles, manuals, patent applications,published patent applications and patents, are expressly incorporatedherein by reference in their entireties.

EXAMPLES Inhibition of T1D and T1D Induced Complications

Materials: Jackson NOD type-1 diabetes female mice, synthetic peptideantigens for generating different endogenous NKA activator antibodies inNOD mice. Method-1: Preparation of active immunization: Specific peptideantigens were synthesized and purified by HPLC. The purified peptideswere sterilized and mixed with TiterMax® adjuvant individually at a 1:1volume ratio and injected (IM) to the mice monthly. Method-2: Detectionof antibody generation: Serum was collected from blood and incubatedwith peptide antigens made against specific binding site for SSA78,SSA401, SSA412, JY2948 and JY421228 antibodies separately. ELISA wasperformed to determine antibody generation. Method-3: Detection of bloodglucose: NOD mice blood glucose was measured weekly using OneTouchsystem. Method-4: Detection of serum insulin concentration: Seruminsulin concentration was determined using Millipore Insulin RIA Kit(RI-13K). Method-5 Detection of Serum creatinine concentration: Serumcreatinine was determined using Creatinine Assay Kit (abcam). Method-6:Detection of NKA activator endogenous antibodies protected pancreatic βcell function and inhibit T1D and T1D complications. FIG. 1 revealshealthy insulin-producing islets in NKA antibody activator-treated NODmice and demonstrate a protective effect of NKA antibody activators onthe structure and function of pancreatic beta cells. FIG. 2 shows SSA412protected beta cell function by promoting beta cell insulin generation.The statistical significant difference between treated and untreatedgroups is p<0.001. FIG. 3 reveals NKA antibody activator-basedimmunotherapy prevented hyperglycemia in type-1 diabetes female NOD mice(p<0.001). FIG. 4 reveals that the group of mice treated with NKAantibody activator SSA412 has a statistically significant longersurvival than untreated group, demonstrating NKA antibody activatorsprolong life span of T1D mice.

Method-6: Detection of NKA activator antibodies prevented T1Dcomplications. FIG. 5 reveals NKA antibody activator-based immunotherapyprotected kidney function and prevented kidney enlargement and failure(n=10). FIG. 6 provide evidence to demonstrate that NKA antibodyactivator protected kidney function and prevented kidney enlargement andfailure (n=10). FIG. 7 demonstrated that the NKA antibodyactivator-based immunotherapy protected kidney function by maintaining anormal creatinine concentration in blood (n=15). (p<0.001). FIG. 8reveals that NKA antibody activator-based immunotherapy protected NKAactivity while NKA activity was significantly impaired in untreateddiabetic kidney (n=15).

What is claimed is:
 1. A method for inhibiting T1D in a subjectcomprising administering an effective amount of an antibody havingbinding specificity for α or β subunit of NKA to a subject in needthereof.
 2. The method of claim 1, wherein the antibody has bindingspecificity for one or more of the peptides represented by SEQ IDNOs:1-7.
 3. The method of claim 1, wherein the antibody is antibodySSA78 or a humanized or human versions thereof, or a fragment orderivative thereof.
 4. The method of claim 1, wherein the antibody isantibody SSA401 or a humanized or human versions thereof, or a fragmentor derivative thereof.
 5. The method of claim 1, wherein the antibody isantibody SSA412 or a humanized or human versions thereof, or a fragmentor derivative thereof.
 6. The method of claim 1, wherein the antibody isantibody JY2948 or a humanized or human versions thereof, or a fragmentor derivative thereof.
 7. The method of claim 1, wherein the antibody isantibody JY421228 or a humanized or human versions thereof, or afragment or derivative thereof.
 8. The method of claim 1, wherein theantibody is in a pharmaceutical formulation comprising the antibody anda pharmaceutically acceptable carrier.
 9. The method of claim 1, whereinthe subject has or is at greater risk than the general population for adisease or condition selected from the group consisting of 1) heart andblood vessel disease and various cardiovascular problems, includingcoronary artery disease with chest pain (angina), heart attack, heartfailure, stroke, atherosclerosis and high blood pressure; 2) nervedamage (neuropathy) in the walls of the blood vessels (capillaries) thatnourish the nerves, especially in the legs causing tingling, numbness,burning or pain, hyperglycemia, lose sense of feeling in the affectedlimbs, nausea, vomiting, diarrhea or constipation; 3) kidney functiondamage (nephropathy), kidney failure or irreversible end-stage kidneydisease; 4) eye damage, damaged the blood vessels of the retina leadingto diabetic retinopathy, cataracts and glaucoma; 5) various footcomplications; 6) skin and mouth infections and Gum diseases; 7)pregnancy complications may lead to miscarriage, stillbirth, birthdefects, diabetic ketoacidosis, retinopathy, pregnancy-induced highblood pressure and preeclampsia or other disease or condition whereininhibition of T1D would be desirable or necessary.
 10. A method forinhibiting and T1D complications in a subject comprising administeringan effective amount of an antibody having binding specificity for the αor β subunit of NKA to a subject in need thereof.
 11. The method ofclaim 8, wherein the antibody has binding specificity for one or more ofthe peptides represented by SEQ ID NOs:1-7.
 12. The method of claim 8,wherein the antibody is antibody SSA78 or a humanized or human versionsthereof, or a fragment or derivative thereof.
 13. The method of claim 8,wherein the antibody is antibody SSA401 or a humanized or human versionsthereof, or a fragment or derivative thereof.
 14. The method of claim 8,wherein the antibody is antibody SSA412 or a humanized or human versionsthereof, or a fragment or derivative thereof.
 15. The method of claim 1,wherein the antibody is antibody JY2948 or a humanized or human versionsthereof, or a fragment or derivative thereof.
 16. The method of claim 1,wherein the antibody is antibody JY421228 or a humanized or humanversions thereof, or a fragment or derivative thereof.
 17. The method ofclaim 10, wherein the antibody is in a pharmaceutical formulationcomprising the antibody and a pharmaceutically acceptable carrier. 18.The method of claim 10, wherein the subject has or is at greater riskthan the general population for a disease or condition selected from thegroup consisting of wherein the subject has or is at greater risk thanthe general population for a disease or condition selected from thegroup consisting of 1) heart and blood vessel disease and variouscardiovascular problems, including coronary artery disease with chestpain (angina), heart attack, heart failure, stroke, atherosclerosis andhigh blood pressure; 2) nerve damage (neuropathy) in the walls of theblood vessels (capillaries) that nourish the nerves, especially in thelegs causing tingling, numbness, burning or pain, hyperglycemia, losesense of feeling in the affected limbs, nausea, vomiting, diarrhea orconstipation; 3) kidney function damage (nephropathy), kidney failure orirreversible end-stage kidney disease; 4) eye damage, damaged the bloodvessels of the retina leading to diabetic retinopathy, cataracts andglaucoma; 5) various foot complications; 6) skin and mouth infectionsand Gum diseases; 7) pregnancy complications may lead to miscarriage,stillbirth, birth defects, diabetic ketoacidosis, retinopathy,pregnancy-induced high blood pressure and preeclampsia or other diseaseor condition wherein inhibition of T1D would be desirable or necessary.19. A method for treating a T1D and T1D complications in a subjectcomprising administering an effective amount of a peptide antigenvaccine for generating antibody having binding specificity for the α orθ subunit of NKA to a subject in need thereof.
 20. The method of claim19, wherein the peptides represented by SEQ ID NOs:1-7 and itsderivatives.
 21. The method of claim 19, wherein the peptide antigen isin a pharmaceutical formulation comprising the peptide antigen and apharmaceutically acceptable carrier.
 22. The method of claim 19, whereinthe disease is selected from the group consisting of 1) heart and bloodvessel disease and various cardiovascular problems, including coronaryartery disease with chest pain (angina), heart attack, heart failure,stroke, atherosclerosis and high blood pressure; 2) nerve damage(neuropathy) in the walls of the blood vessels (capillaries) thatnourish the nerves, especially in the legs causing tingling, numbness,burning or pain, hyperglycemia, lose sense of feeling in the affectedlimbs, nausea, vomiting, diarrhea or constipation; 3) kidney functiondamage (nephropathy), kidney failure or irreversible end-stage kidneydisease; 4) eye damage, damaged the blood vessels of the retina leadingto diabetic retinopathy, cataracts and glaucoma; 5) various footcomplications; 6) skin and mouth infections and Gum diseases; 7)pregnancy complications may lead to miscarriage, stillbirth, birthdefects, diabetic ketoacidosis, retinopathy, pregnancy-induced highblood pressure and preeclampsia or other disease or condition whereininhibition of T1D would be desirable or necessary.